The primary goal of this research is to elucidate the molecular mechanisms involved in two types of regulation of amino acid biosynthesis in bacteria. The first type of mechanism is gene or operon specific and regulates amino acid biosynthesis with respect to the cell's need for a particular amino acid. In the case of histidine biosynthesis, which we are studying as a model system, the cell senses histidine sufficiency or deficiency through the amount of charged tRNAHis. In addition, charged tRNAHis must contain two pseudouridine base-modifications in the anticodon loop in order to repress the histidine operon. In general, the regulatory mechanism through which charged/modified tRNAHis acts now appears to involve translational-control-of-transcription-termination in the regulatory region of the operon. This is quite different than other bacterial genetic regulatory mechanisms that involve binding of regulatory proteins (repressors or activators) to specific control sites in DNA. We would like to understand precisely how the translational control aspect of the mechanism works and how the pseudouridine tRNA modification modulates translation. Both translational control and modified bases in tRNA are likely to be very important in higher-cell gene regulation. The second type of mechanism involves the unusual nucleotide guanosine 5'-diphosphate 3'-diphosphate (ppGpp). This molecule appears to be part of a super-control system that adjusts many cellular processes in response to the cell's need for amino acids in general. Specifically, we will attempt to identify and characterize the components with which ppGpp interacts to increase transcription initiations of the histidine biosynthetic, and lactose catabolic, operons. These mechanisms of specific and general amino acid regulation are being pursued through use of genetic-physiological techniques and cell-free systems that measure transcription and translation as indicators of gene regulation.